populations of
Maytenus Ilicifolia
Mart. ex Reiss
Mossi, AJ.
a*, Cansian, RL.
a, Leontiev-Orlov, O.
a, Cechet, JL.
a,
Carvalho, AZ.
a, Toniazzo, G.
aand Echeverrigaray, S.
baDepartamento de Ciências Agrárias, Campus de Erechim, Universidade Regional Integrada do Alto Uruguai e das Missões – URI,
Av. 7 de setembro, 1621, CEP 99700-000, Erechim, RS, Brazil bInstituto de Biotecnologia, Universidade de Caxias do Sul – UCS, Av. Francisco Getúlio Vargas, 1130, CEP 95070-560, Caxias do Sul, RS, Brazil Received February 27, 2008 – Accepted May 20, 2008 – Distributed May 31, 2009
*e-mail: amossi@uricer.edu.br (With 5 figures)
Abstract
The aim of this work was to analyze genetic variability in 18 populations of Maytenus ilicifolia, and representatives of
Maytenus aquifolia and Maytenus evonymoidis, collected in the states of Mato Grosso do Sul, Paraná, Santa Catarina and Rio Grande do Sul, using RAPD molecular markers. Considering total samples of the three species, 263 ampli-fied fragments were identiampli-fied, of which 72.2% showed to be polymorphous. The index of similarity (Jaccard coef-ficient) was on average 0.64 between M. ilicifolia and M. aquifolia; 0.47 between M. ilicifolia and M. evonymoidis; and 0.44 between M. aquifolia and M. evonymoidis. The analysis of groupings by the UPGMA algorithm allowed to clearly separate the three analyzed species. In determining the variability in M. ilicifolia, 222 bands were identified, on average 11.1 bands per primer, being 43.2% polymorphous. The index of similarity (Jaccard coefficient) in the bulks of each population in M. ilicifolia was, on average, 0.92 and the index of similarities among the populations was 0.83. The analysis of groupings with the UPGMA algorithm and the analysis of the main coordination (PCO), allowed the separation of the analyzed populations into three groups, the populations from the south of Rio Grande do Sul and the population from Mato Grosso do Sul standing out. A relation between the groupings found and the edaphoclimatic conditions of the collecting places was observed.
Keywords: conservation genetics, Espinheira Santa, native populations, molecular markers.
Diversidade genética de conservação em populações
nativas de
Maytenus ilicifolia
Mart. ex Reiss
Resumo
O objetivo deste trabalho foi analisar a variabilidade genética em dezoito populações de Maytenus ilicifolia, e re-presentantes de Maytenus aquifolia e Maytenus evonymoidis, coletadas nos Estados do Mato Grosso do Sul, Paraná, Santa Catarina e Rio Grande do Sul, utilizando marcadores moleculares RAPD. Considerados todos os representantes das três espécies, foram identificados 263 fragmentos amplificados, dos quais 72,2% mostraram-se polimórficos. O índice de similaridade (coeficiente de Jaccard) foi em média de 0,64 entre M. ilicifolia e M. aquifolia, de 0,47 entre
M. ilicifolia e M. evonymoidis e de 0,44 entre M. aquifolia e M. evonymoidis. A análise de agrupamentos através do algoritmo UPGMA permitiu separar claramente as três espécies analisadas. Na determinação da variabilidade dentro de M. ilicifolia foram identificadas 222 bandas, em média de 11,1 bandas por primer, sendo 43,2% polimórficas. O índice de similaridade (coeficiente de Jaccard) dentro dos bulks de cada população em M. ilicifolia foi em média de 0,92, e índices de similaridade entre as populações de 0,83. A análise de agrupamentos através do algoritmo UPGMA e análise de coordenadas principais (PCO), permitiram separar as populações analisadas em três grupos, destacando as populações do sul do RS e a de MS das outras avaliadas. Foi observada uma relação entre os agrupamentos encon-trados e as características edafoclimáticas dos locais de coleta.
1. Introduction
The use of medicinal plants in Brazil is intense, espe-cially by populations of a lower income, since Brazilian flora displays great biodiversity. However, scant knowl-edge and the bad use of natural resources have led to the loss of populations, and several medicinal plants are included in the list of species threatened with extinction. The genus Maytenus belongs to the Celastraceae family, and several of its species are used in popular medicine in different regions of the world. The great use of Maytenus ilicifolia Mart. ex Reiss 1861 has increased the degrada-tion of this species, and thus it is presently included in FAO’s list for priority species for studying and conserva-tion in South America. In this context, this work aimed to contribute with studies that help towards the conservation of this species.
In Brazil, the species Maytenus aquifolia Mart. 1841 and Maytenus ilicifolia are popularly used as anti-spasmodic, contraceptive, anti-ulcerous, diuretic, cicatrizing, and analgesic features. Studies performed with animals in the laboratory (Oliveira et al., 1991; Souza-Formigoni et al., 1991) proved the anti-ulcerous effect of M. aquifolia and M. ilicifolia, which was at-tributed to the increasing of volume and pH of the gastric juices (Souza-Formigoni et al., 1991), and their activities as an anti-oxidant (Vellosa et al., 2006), and anti septic and cicatrizing effects (Carlini and Braz, 1998). Several compounds are presented in the extract, such as phenols, tannins and terpenes (Camparoto et al., 2002).
Carvalho-Okano (1992) identified the southwest region as the primary centre of specific diversity of
Maytenus in Brazil, because it houses the biggest number of species. However, the current geographical distribu-tion of M. ilicifolia occurs principally in the southern region of Brazil (Paraná, Santa Catarina and Rio Grande do Sul), being preferably found in the sub-woods or the margins of rivers. The author also mentions the fact that she did not find material in herbariums in São Paulo and Mato Grosso do Sul, where the occurrence would be rare.
The low frequency of occurrence, the intense use in phitotherapy by the population and the big anthropic ac-tion in the region of natural occurrence of this species has taken it to degrading. In this sense, genetic studies are fundamental for the handling and conservation of this species.
The use of molecular markers is a powerful tool in the genetic study of populations, RAPD (Random Amplifies Polymorphic DNA) being suitable for the analysis of ge-netic diversity in natural populations of allogamous spe-cies (Ferreira and Grattapaglia, 1998). Few studies on genetic and chemical diversity have been performed in
M. ilicifolia so far. Bittencourt (2000) studied the genetic variability of two populations of M. ilicifolia in the state of Paraná, using RAPD molecular markers, and Perecin (2002), using biochemical markers (allozymes), studied genetic diversity in five populations of M. aquifolia in
the state of São Paulo and one population of M. ilicifolia
in Santa Catarina.
The objective of the present work was to analyze ge-netic variability in 18 native populations of M. ilicifolia
distributed in the south and mid-west regions of Brazil (Mato Grosso do Sul, Paraná, Santa Catarina and Rio Grande do Sul), using RAPD molecular markers, aiming to contribute for determining strategies for the species’ conservation.
2. Material and Methods
Plants of the eighteen native populations of espinhei-ra santa (M. ilicifolia) were sampled in three Brazilian states: Mato Grosso do Sul, in the town of Ponta Porã; Paraná, in the towns of Lapa, Irati, Guarapuava and Mangueirinha; Santa Catarina, in the towns of Lages, São Joaquim and Caçador; and Rio Grande do Sul, in the towns of Santana do Livramento, Canguçu, Unistalda, Vale Verde, Soledade, Flores da Cunha, Bom Jesus, Erechim, Barão de Cotegipe and São José (Figure 1). The group of plants geographically isolated was consid-ered as a population. Thirty adult plants collected ran-domly in each area represented each population, forming 3 bulks of 10 individuals for each population. The mini-mum distance between two plants was 10 m. Besides genetic analysis in M. ilicifolia populations, M. aquifolia
and Maytenus evonymoides Reissekwere used for inter-specific comparison.
The sample regions were based on Koppen’s clas-sification, based on environmental characteristics (lati-tude, longi(lati-tude, vegetation, annual average temperature, climate, geomorphology, and types of soil).
2.1. Genetic analysis
The leaves collected in each plant were immediately placed in liquid nitrogen, and then stored in a freezer at –80 °C until DNA extraction, which occurred with the formation of three bulks with 10 plants per population.
For the isolation of total DNA, the method de-scribed by Doyle and Doyle (1987), modified for the use of M. ilicifolia (Bittencourt, 2000) was used. The basic process consists of: maceration of about 150 mg of leaves in liquid nitrogen; addition of 750 µL of ex-traction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl pH 8.0, 0.2% 2-Mercaptoetanol, 0.01% Proteinase K, 1% PVP); maintenance in immer-sion for 45 minutes at 65 °C; removing residual protein with one volume of 24:1 chloroform: isoamyl alcohol un-til total cleaning of DNA; precipitation with 2/3 volume of isopropanol and two washings with 1000 µL ethanol 70%; re-suspension in 150 µL of TE (Trisma: EDTA – 10:1); quantification in UV spectrophometry at 260 nm and confirmation of integrity and purity in spectrophom-etry UV at 280 nm and in 0.8% agarose gel.
(50 mM Tris-HCl pH 9.0; 50 mM KCl), dNTPs (200 mM of each), 0.2 mM of primer, 3 mM of MgCl2, 0.25 mM of TRITON and 1.5 U of Taq DNA polimerase Gibco BRL (Life Technologies, São Paulo, Brazil) and 40-50 ng of DNA.
For the selection of the primers, kits OPA, OPB, OPD, OPF, OPH, OPW, and OPY from Operon Technologies, containing 20 primers each were used, aiming to iden-tify those which presented better results for M. ilicifolia, evaluating the quantity of bands produced, their intensi-ties, their repetition and the polymorphism generated by them.
The amplification was performed in thermocycler (model PTC 100, MJ Research INC., Watertown, MA). The process of amplification was based on the following sequence: 3 minutes at 92 °C and 40 cycles of 1 minute at 92 °C, 1 minute at 35 °C and 2 minutes at 72 °C. Then, 3 minutes at 72 °C and cooling at 4 °C until the retreat of the samples.
Electrophoretic separation was performed in agarose gel 1.4% in buffer TBE lX (0.089 M Trisma, 0.089 M Boric Acid and 0.008 M EDTA) in horizontal electro-phoresis, and a potential of 90 V. DNA Ladder 100 bp from Gibco (BRL) was used as a molecular weight marker. The visualization of fragments was done with ethidium bromide and the observation was performed using UV light. The gels were photographed using the digital CEL-PRO System (Media Cybernetics, Silver Spring, MD). For each primer analyzed, three gels
con-taining a bulk of each population of M. ilicifolia were prepared together with one representative from the other two species.
The data analysis was performed as following: in de-termining genetic variability, the data obtained through the determination of presence or absence of bands formed a matrix, which was analyzed with the help of a statistic pack (MVSP). The dendograms were built by algorithm UPGMA (Unweighted Pair Group Method Using Arithmetic Averages), developed by Sokal and Michener (1958) using Jaccard coefficient of similar-ity. Confidence limits for groupings were calculated by randoming 100 samples of the results using the Winboot program (Yap and Nelson, 1996). The analysis of the main intra and inter-specific coordination was analyzed by MVSP (Multi-Variate Statiltical Package 3.1, Kovach Computing Service).
3. Results and Discussion
In the study of variability, 20 primers from Operon Technologies (Table 1) were used. The primers used were selected from 140 decanumeric primers from kits OPA, OPB, OPD, OPF, OPH, OPW and OPY. The se-lection of primers was based on quantity, intensity and repetition of amplified fragments and results obtained by Bittencourt (2000). Considering that the amplification of fragments can be affected by factors such as concen-tration of reaction components and different conditions
Ponta Porã Ponta Porã
Santana do Livramento Santana do Livramento
Unistalda Unistalda Lages Lages Soledade Soledade São Joaquim São Joaquim São José São José Caçador Caçador Mangueirinha Mangueirinha Irati Irati Guarapuava Guarapuava Canguçu Canguçu Maringá
Maringá LondrinaLondrina Concepción Concepción Paraguay Paraguay Asunción Asunción Pilar Pilar Corrientes Corrientes Paraná River Paraná River Ypoa Lake Ypoa Lake San Rafael San Rafael Yuty Yuty Posadas Posadas Serra de Amambai Serra de Amambai Uruguay Ri ver Uruguay Ri ver Iguaçu National ParkIguaçu National Park
Foz do Iguaçu Foz do Iguaçu
Cascavel Cascavel
Caxias do Sul Caxias do Sul Paraná Paraná Brazil Brazil São Paulo São Paulo São Paulo São Paulo Santo André Santo André Rio Claro Rio Claro Campinas Campinas Jundiaí Jundiaí Osasco Osasco Embu Embu São Vicente São Vicente SantosSantos
Ciudad del Este Ciudad del Este Pelotas Pelotas Porto Alegre Porto AlegreSão LeopoldoSão Leopoldo Santa Maria
Santa Maria Rio Grande do Sul Rio Grande do Sul
Passo Fundo Passo Fundo Itajaí Itajaí Florianópolis Florianópolis Joinville Joinville Ponta Grossa Ponta Grossa Pilcomayo Ri ver Pilcomayo Ri ver Uruguay Uruguay Rivera Rivera 17 17 6 6 2 2 8 8 11 11 5 5 1515
12 12 9 9 13 13 7 7 18 10 10 4 4 3 3 South America South America Vale Verde Vale Verde Erechim Erechim São Joaquim National ParkSão Joaquim National Park
Serra do MarSerra do Mar
Serra Geral Serra Geral Mato Grosso do Sul
Mato Grosso do Sul
Curitiba Curitiba Lapa Lapa Santa Catarina Santa Catarina Argentina Argentina 16 16
0 100 200 300 400 km
Negro River Negro River Iguazu Ri ver Iguazu Ri ver Barão de CotegipeBarão de Cotegipe
1 14
Figure 1. Collecting places of the 18 populations of M. ilicifolia in the states of Mato Grosso do Sul, Santa Catarina, Paraná
of amplification cycles (Geburek, 1997), only amplified fragments that presented a high intensity and reproduc-tion in three repetireproduc-tions of amplificareproduc-tion were considered for the evaluation.
In the analysis of the bulks representative of each of the 18 populations of M. ilicifolia, M. aquifolia and
M. evonymoidis, 263 fragments were considered, be-ing 190 (72.2%) polymorphics and 73 (27.8%) mono-morphics. The amplified fragments presented between 100 and 2200 bp. The average number of fragments per primer was 13.15, and this result was found to be supe-rior to the one found in the intra-population analysis in
M. ilicifolia of 10.9 (Mossi et al., 2007). This difference can be attributed to the presence of 41 (15.6%) bands from M. aquifolia and M. evonymoidis. The average number of bands obtained per primer is superior to the one found by Bittencourt (2000) in M. ilicifolia, which was 7.42 per primer. This difference can be attributed to distinct laboratorial conditions, to the primers used, and to the rigidity established in the choice of fragments for analysis.
Regarding the presence or absence of bands in the species, from the 263 bands analyzed, 49 bands (18.6%) were observed to occur only in M. ilicifolia, nine bands (3.4%) in M. aquifolia and 27 (10.3%) in M. evonymoidis.
M. ilicifolia and M. aquifolia are both widely used in popular medicine, and they are often confused due to their morphologic similarities (Carvalho-Okano, 1992).
The low number of specific bands for M. aquifolia con-firms this proximity, but it also brings the possibility of using molecular markers in the identification of mixtures and adulteration.
The index of similarity (Jaccard coefficient) between
M. ilicifolia and M. aquifolia varied between 0.59 and 0.69 with average of 0.64, between M. ilicifolia and
M. evonymoidis it was from 0.43 to 0.50 with an average of 0.47, and between M. aquifolia and M. Evonymoidis
it was on average 0.44. As expected, the indices of simi-larities between the species were inferior in comparison to the ones within the species (0.72 to 0.79) (Mossi et al., 2007).
The analysis of groupings by means of the UPGMA algorithm (Figure 2) allowed to clearly separate the species analyzed with 100% of confidence, being
M. evenymoidis the most distant one. The high presence of sharing fragments between M. ilicifolia and M. aquifolia
indicates a high relationship between the species. In the same way, Perecin and Kageyama (2002), in studies with allozymes loci, verified the presence of many al-leles shared between M. ilicifolia and M. aquifolia, and the authors suggest the possibility that the species are not completely isolated reproductively or that this fact occurred in a not very distant space of time.
In M. ilicifolia, 222 bands from 20 primers, which originated, on average, 11.1 bands per primer were ana-lyzed. From this total, 126 (56.8%) were monomorphics
Table 1. Total number and number of polymorphic fragments obtained in each primer used in the three populations
ana-lyzed.
Primer Sequence
(5’ to 3’)
Total fragments Specific fragments
M. ilic M. aqui M. evon Poli M. ilic M. aqui M. evon
OPA-04 CAGGCCCTTC 12 6 8 12 1 1
-OPA-08 GTGACGTAGG 8 6 6 11 3 1 3
OPA-09 GGGTAACGCC 9 5 4 8 3 - 1
OPA-11 CAATCGCCGT 16 11 10 11 4 - 1
OPA-19 CAAACGTCGG 9 4 8 9 3 1 2
OPB-07 GGTGACGCAG 13 9 9 12 3 1 2
OPB-11 GTAGACCCGT 15 9 10 14 4 - 1
OPB-12 CCTTGACGCA 8 4 7 10 1 1 2
OPB-13 TTCCCCCGCT 6 4 2 4 1 -
-OPD-20 ACCCGGTCAC 13 10 11 9 2 - 1
OPD-08 GTGTTGCCCA 13 13 9 8 2 -
-OPF-01 ACGGATCCTG 16 9 10 13 6 2 1
OPF-10 GGAAGCTTGG 11 10 8 11 1 - 4
OPH-03 AGACGTCCAC 12 9 9 7 2 - 2
OPW-08 GACTGCCTCT 8 6 6 6 3 1 1
OPW-16 CAGCCTACCA 9 7 5 12 2 - 4
OPY-08 AGGCAGAGCA 9 7 7 8 2 1 2
OPY-10 CAAACGTGGG 9 4 6 7 2 -
-OPY-13 GGGTCTCGGT 13 8 12 7 1 -
-OPY-18 GTGGAGTCAG 13 10 8 11 3 - 2
and 96 (43.2%) polimorphics. The level of polimorphism was lower than that observed by Mossi et al. (2007), who evaluated the intra and inter population genetic variabili-ty of M. ilicifolia. In this study a polymorphism of 71.5% was observed.
The index of similarity (Jaccard coefficient) inside the bulks of each population in M. ilicifolia varied be-tween 0.87 and 0.96 with an average of 0.92 and the in-dices of similarity between populations varied from 0.83 to 0.91 with an average of 0.83.
The analysis of grouping using the UPGMA algo-rithm (Figure 3) allowed the characterization of each population, since the internal bulks remained next, grouping themselves with indices of average confidence of 88%. Group I was formed of a population derived from Ponta Porã (MS), group II by populations from Santana do Livramento, Vale Verde, Canguçu and Unistalda, all
M. evonymoidis M. aquifolia Ponta Porã
Erechim
Irati São Joaquim
Jaccard’s coefficient
01 03 02 01 03 02 01 03 02 01 02 03 01 02 03 01 02 03
0.4 0.5 0.6 0.7 0.8 0.9 1.0
Figure 2. Dendogram of some populations of M. ilicifolia
and the species M. aquifolia and M. evonymoidis deter-mined by RAPD, using Jaccard’s coefficient and UPGMA grouping.
01 02 03 01 03 02 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 03 02 01 02 03 01 03 02 01 03 02 01 02 03 01 02 03 01 02 03 01 03 02 01 03 02 01 02 03 01 02 03
0.76 0.80 0.84 0.88 0.92 0.96 1.00
S. do Livramento Ponta Porã
B. Jesus S. Joaquim S. José Lages Caçador Soledade Guarapuava Erechim B. de Cotegipe Irati
F. de Cunha Mangueirinha Lapa Canguçu Vale Verde Unistalda
UPGMA (Jaccard’s coefficient)
Figure 3. Dendrogram based on the analysis of grouping (UPGMA) of genetic similarity estimation (Jaccard’s coefficient)
of them from the southern region of Rio Grande do Sul, and group III by the thirteen remaining populations. The separation of these three groups was confirmed by the analysis of the main coordinators (PCO), using Euclidian distances (Figure 4) in which a tendency of separation between Ponta Porã’s population and populations from the southern region of Rio Grande do Sul is observed.
Figure 5 shows the comparison of the groupings obtained by the analysis of molecular markers and the groupings of the regions where the plants were sampled based on Koppen’s classification. It may be noted that the group separation corresponds to different environ-ments.
The high similarities among populations and the low definition of the groups corroborate with the results ob-tained by Bittencourt (2000). This might be related to
the fact that the southwest region is the primary centre of specific diversity of Maytenus sp., and that M. ilicifolia
occurs preferably in the sub-woods of Araucaria Forest and on the margins of rivers (Carvalho-Okano, 1992), and in the arboreal groupings in regions of steppes. The Araucaria forest has a recent origin (2,500 to 4,000 years) in the southern region of Brazil (Joly et al., 1999), and thus it has a recent occurrence of species related to it.
Despite the genetic proximity, one can notice a ten-dency of distance and isolation of Ponta Porã’s popula-tion, possibly influenced by a founder effect and kept by its relative isolation from the other regions of occur-rence of the species. In the southern region, there is also a tendency of grouping and distancing of Santana do Livramento, Canguçu, Unistalda and Vila Verde’s popu-lations, possibly due to the pressure of selection associ-ated to climatic differences and vegetation. Considering the dispersion centre of the species, the southern region corresponds to a marginal area of distribution, with oc-cupation of new environments, which normally brings about variation of allelic frequencies.
The results allowed us to conclude that RAPD molecular markers are effective in the differentiation of the studied species (M. ilicifolia, M. aquifolia and
M. evonymoidis) and in the study of genetic variability in M. ilicifolia.
The molecular markers showed a high genetic simi-larity in M. ilicifolia’s populations. However, they also allowed the separation of populations in groups of simi-larity, which are related to the distinct environments of occurrence of M. ilicifolia (Southern Brazil, Araucaria forest and the mountain ridges of Mato Grosso do Sul).
PCO (Euclidean distance)
Axis 2 Axis 3
Axis 1 –0.8 –1.6 –2.4 –3.2 –3.9 –0.8 –1.6 –2.4 –3.2 –3.9 0.8 1.6 2.4 3.2
0.8 1.6 2.4 3.2
0.8 1.6 2.4 3.2 0.8 1.6 2.4 3.2
Ponta Porã
Vale Verde Unistalda
Canguçu and Santana do Livramento
–0.8 –1.6 –2.4 –3.2
–3.9 –3.2 –2.4–1.6 –0.8 –3.9
Figure 4. Analysis of Main Coordination by Euclidian
dis-tance of the 18 populations of M. ilicifolia analyzed.
Figure 5. UPGMA grouping with Euclidian distances of the 18 populations analyzed, based on environmental characteristics
of each place (latitude, longitude, altitude, annual average temperature, vegetation, Koppen climatic classification, geomor-phology and kinds of soil).
Euclidean distance
Soledade Unistalda Santana do Livramento Vale Verde Canguçu Ponta Porã Lapa Irati Flores da Cunha São Joaquim Lages Caçador Bom Jesus Guarapuava Mangueirinha São José Erechim Barão de Cotegipe
This study suggests that the formation of three distinct groups in Brazil is occurring possibly due to the influ-ence of evolutionary forces, since the groups are sepa-rating themselves according to environmental conditions (Koppen’s classification).
However, in the last 50 years, a great anthropogenic impact is occurring in these regions, with intense frag-mentation that caused the loss of populations and the consequent isolation of the remaining ones.
In species that become fragmented into small popula-tions and isolated subpopulapopula-tions, the random change in gene frequencies (genetic drift) and the mating of related individuals (biperental inbreeding) have driven species to the brink of extinction (Geburek, 1997). The challenge now is to develop good conservation programs and to put them into action. Romeiras et al. (2007) concluded that the actions for conservation of Echium stenosiphon
should be based on the genetic diversity of the groups identified in the population. In the same way, we can propose that the conservation programs of M. ilicifolia
must consider the genetic variability of the three distinct groups detected by RAPD analysis.
Acknowledgements — The authors thank CNPq, SCT-RS, and FAPERGS for the financial support, and EMATER/RS and EMBRAPA/Florestas for technical assistance.
References
BITTENCOURT, JVM., 2000. Variabilidade genética em populações naturais de maytenus ilicifolia por meio de marcadores RAPD. Curitiba: Universidade Federal do Paraná. 58p. Dissertação de Mestrado.
CAMPAROTO, ML., TEIXEIRA, RO. and MANTOVANI, MS., 2002. Effects of Maytenus ilicifolia Mart. and Bauhinia candicans Benth infusions on onion root-tip and rat bone-marrow cell. Genetics and Molecular Biology, vol. 25, no. 1, p. 85-89.
CARLINI, EA. and BRAZ, S., 1998. Efeito protetor do liofilizado obtido do abafado de Maytenus sp. (Espinheira-Santa) contra úlcera gástrica experimental em ratos. In CARLINI, E.A. (Eds.). Estudo da ação antiúlcera gástrica de plantas brasileiras. Brasília: Central de Medicamentos - CEME/AFIP. p. 21-35.
CARVALHO-OKANO, RM., 1992. Estudos taxonômicos do gênero Maytenus MOL. emend. Mol. (Celastraceae) do Brasil Extra-amazônico. Campinas: Universidade Estadual de Campinas. 252p. Tese de Doutorado.
DOYLE, JJ. and DOYLE, JL., 1987. A rapid DNA isolation method for small quantities of fresh tissues. Phytochemical Buletin, vol. 19, p. 11-15.
FERREIRA, EM. and GRATTAPAGLIA D., 1998. Introdução ao uso de marcadores moleculares em análise genética. Brasília: EMBRAPA; CENARGEN.
GEBUREK, T., 1997. Isozymes and DNA markers in gene conservation of forest trees. Biodiversity and Conservation, vol. 6, no. 12, p. 1639-1654.
JOLY, CA., AIDAR, MPM., KLINK, CA., McGRATH, DG., MOREIRA, AG., MOUTINHO, P., NEPSTAD, DC., OLIVEIRA, AA., POTT, A., RODAL, MJN. and SAMPAIO, EVSB., 1999. Evolution of the Brazilian phytogeography classification systems: implications for biodiversity conservation. Ciência e Cultura, vol. 51, no. 5-6, p. 331-348.
MOSSI, AJ., CANSIAN, RL., LEONTIEV-ORLOV, O., ZANIN, EM., OLIVEIRA, CH.; CECHET, ML., CARVALHO, AZ. and ECHEVERRIGARAY, S., 2007. Intra and inter populational genetic variability in Maytenus ilicifolia Mart. Ex Reiss, through RAPD Markers. Revista Brasileira de Biologia = Brazilian Journal of Biology, vol. 67, no. 4, p. 957-961. OLIVEIRA, MGM., MONTEIRO, MG., MACAUBAS, C., BARBOSA, VP. and CARLINI, EA., 1991. Pharmacologic and toxicologic effects of two Maytenus species in laboratory animals. Journal of Ethnopharmacology, vol. 34, no. 1, p. 29-41.
PERECIN, MB. and KAGEYAMA, PY., 2002. Variabilidade isoenzimática em populações naturais de Espinheira-Santa Maytenus aquifolia Mart. e Maytenus ilicifolia Mart. ex Reiss e suas implicações para o manejo da conservação. Revista Brasileira Planta Médica, vol. 4, no. 2, p. 80-90.
ROMEIRAS, MR., COTRIM, HC., DUARTE MC. and PAIS, MS., 2007. Genetic diversity of three endangered species of Echium L. (Boraginaceae) endemic to Cape Verde Islands. Biodiversity and Conservation, vol. 16, no. 2, p. 547-566. SOKAL, RR. and MICHENER, CD., 1958. A statistical method for evaluating systematic relationships. The University of Kansas Scientific Bulletin, vol. 38, p. 1409-1438.
SOUZA-FORMIGONI, ML., OLIVEIRA, MGM., MONTEIRO, MG., SILVEIRA-FILHO, NG., BRAZ, S. and CARLINI, EA., 1991. Antiulcerogenic effects of two Maytenus species in laboratory animals. Journal of Ethnopharmacology, vol. 34, no. 1, p. 21-27.
VELLOSA, JCR., KHALIL, NM., FORMENTON, VAF., XIMENES, VF., FONSECA, LM., FURLAN, M., BRUNETTI, IL. and OLIVEIRA, OMMF., 2006. Antioxidant activity of Maytenus ilicifolia root bark. Fitorepia, vol. 77, no. 3, p. 243-244.
WILLIAMS, JGK., KUBELIK, AR., LIVAK, KJ., RAFALSKI, LA. and TINGEY, SV., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic acids research, vol. 18, no. 22, p. 6531-6535.
